Masticatory and spontaneous pain associated with temporomandibular joint disorders (TMJD) is a prevalent and significant contributor to orofacial pain. TMJD pain presents for too many patients as a debilitating chronic trigeminal pain condition that impacts their lives overwhelmingly. TMJD pain is undoubtedly a serious unmet medical need. Unfortunately, current treatments for TMJD pain are lacking in effectiveness, primarily due to: 1) shortcomings of current TMJD preclinical models to faithfully model patients? cardinal complaints; 2) the elusiveness of the molecular, cellular and neural-circuit mechanisms that underlie TMJD pain. Our proposal seeks to address both key issues. TRP channels relevant for pain, pain-TRPs, expressed by trigeminal ganglion (TG) sensory neurons, have been critically implicated in both acute and chronic pain and represent possible bona-fide targets for development of rationally-guided anti-pain strategies. However, their direct-mechanistic roles in masticatory and spontaneous pain of TMJD are elusive and in need of clarification. In our previously published and preliminary studies, we developed and validated bite force and conditioned place preference assays for measuring masticatory and spontaneous pain, induced by inflammatory injury to the TMJ or ligation of the tendon of the masseter muscle, both methods evoking long- lasting pain-behavior. Using bite force metrics, we found TMJ inflammation-induced masticatory pain to be significantly attenuated, but not fully reversed in Trpv4 knockout (KO) mice, suggesting that the residual pain might be mediated by other pain-TRPs. Our gene expression studies demonstrated that TRPV1 and TRPA1 were up-regulated in the TG in response to TMJ inflammation, moreover, in a Trpv4-dependent manner. Therefore, we hypothesize that TRPV1 and TRPA1, like TRPV4, contribute to TMJD pain, in particular via the ensemble of their signaling. This hypothesis will be examined using our two models of TMJ injury. Pain- behavior read-out metrics will be bite force and conditioned place preference. We will employ a combination of pharmacologic and genetic loss-of-function approaches for the targeted genes as well as selective neuronal silencing using specific TRP agonists combined with positively charged lidocaine derivative (QX314). Also, we will target TMEM100, a recently discovered adaptor of TRPV1-TRPA1 functional interactions, by using a TMEM100-inhibitory peptide for local injections and sensory neuron-TMEM100 conditional knockout mice. Our three Specific Aims will examine the contribution of TRPV1, TRPV4, TRPA1 and TMEM100 to pathogenesis of TMJD pathologic pain including assessment of neurogenic inflammation, a pathogenic mechanism for which we have collected exciting pilot data revealing its key contribution to TMJD pain. Successfully addressing our ambitious, yet feasible Aims will provide a rich yield of new fundamental insights and also readily translatable new knowledge. These will empower us to overcome a serious unmet medical need that our society faces in the age of the ?opioid epidemic?, namely chronic TMJD pain, by applying a rationally-guided new strategy.